CN110300600A - Utilize the combined therapy of antibody-drug conjugates and PARP inhibitor - Google Patents

Abstract

The present invention provides a kind of methods of cancer for treating subject, and the method includes applying the antibody-drug conjugates (ADC) and a effective amount of poly- ADP ribose polymerase (PARP) inhibitor that a effective amount of CD33 is targeted to the subject.Additionally provide the pharmaceutical composition of the ADC comprising a effective amount of CD33 targeting and a effective amount of PARP inhibitor.

Description

Combination therapy with antibody-drug conjugates and PARP inhibitors

Related applications

This application claims priority to US Provisional Patent Application Serial No. 62/416,383, filed November 2, 2016, the entire contents of which are expressly incorporated herein by reference.

sequence listing

This application contains a Sequence Listing that has been electronically filed in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on October 26, 2017 is named 121162-03720_SL.txt and is 27,280 bytes in size.

Background of the Invention

Acute myeloid leukemia (AML) is associated with the accumulation of abnormal blast cells in the bone marrow. Acute myeloid leukemia (AML) is one of the most common types of leukemia in adults. In the United States alone, more than 18,000 new cases of AML are identified each year, and more than 10,000 deaths are associated with AML. Despite high initial response rates to chemotherapy, many patients with acute myeloid leukemia (AML) fail to achieve complete remission. In fact, most patients with AML relapse within 3-5 years from diagnosis.

The leukocyte differentiation antigen CD33 is a 364 amino acid transmembrane glycoprotein with sequence homology to members of the sialadhesin family, including myelin-associated glycoprotein and CD22, as well as sialadhesin itself (S. Peiper, 2002, Leucocyte Typing VII, White Cell Differentiation, Antigens, Proceedings of the Seventh International Workshop and Conference, Oxford University Press, p. 777).

The expression of CD33 appears to be highly specific to the hematopoietic compartment, with strong expression on myeloid precursor cells (S. Peiper, 2002). It consists of myeloid progenitor cells (eg CFU-GEMM, CFU-GM, CFU-G and BFU-E), monocytes/macrophages, granulocyte precursors (eg promyelocytes and myeloid cells, but is Upon differentiation, expression decreases) and mature granulocytes (but at lower levels) express (S. Peiper, 2002). Anti-CD33 monoclonal antibodies have shown that CD33 is expressed by clonally formed acute myeloid leukemia (AML) cells in greater than 80% of human cases (LaRussa, V.F. et al., 1992, Exp. Hematol. 20:442-448). In contrast, "primitive colonies" are generated in vitro (Leary, A.G. et al., 1987, Blood 69:953) and induce hematopoietic long-term bone marrow cultures (Andrews R.G. et al., 1989, J. Exp. Med. 169:1721; Sutherland, H.J. et al., 1989, Blood 74:1563) pluripotent hematopoietic stem cells appear to lack CD33 expression.

Due to the selective expression of CD33, antibody drug conjugates (hereinafter referred to as "ADCs") that combine cytotoxic drugs with monoclonal antibodies that specifically recognize and bind CD33 have been proposed for selective targeting of AML cells. The therapy is expected to leave stem cells and primitive hematopoietic progenitor cells unaffected. Recently, a CD33-targeted ADC utilizing a novel DNA alkylating agent DGN462 was reported comprising an indolinyl-benzodiazepine dimer containing a monoimine moiety (see, eg, U.S. Patent No. 8,765,740, 8,889,669, 9,169,272 and 9,434,748), which showed anticancer activity in in vitro and in vivo hematological cancer models. Although such ADCs show great promise, there is still a need for improved methods of using ADCs to treat patients with cancer, especially hematological cancers such as AML.

SUMMARY OF THE INVENTION

Poly-ADP ribose polymerase (PARP) inhibitors are known for the treatment of solid tumors, especially those with DNA repair deficiencies. Its efficacy in the treatment of hematological cancers has not been fully established. It has now surprisingly been found that the combination of a CD33-targeted ADC containing an indolinyl-benzodiazepine dimer cytotoxic payload with a PARP inhibitor has In vitro and in vivo leukemia cells have a synergistic effect. For example, when human CD33+ acute myeloid leukemia cells (HEL, MV4-11 and HL60) were treated with a combination of the CD33-targeted ADC IMGN779 and the PARP inhibitor olaparib, an increase in cancer cell proliferation was observed. Synergistic reduction (see Example 1). Additionally, the combination of IMGN779 with olaparib i) further reduced tumor burden in an acute myeloid leukemia xenograft animal model compared to the drug alone (see Example 2); Colony formation of primary cells from patients with relapsed/refractory acute myeloid leukemia characterized by a complex karyotype or FLT-3 mutation (see Example 3). Based on these surprising findings, the present invention provides the use of indolinyl-benzodiazepine dimer cytotoxic payload CD33-targeted ADCs in combination with the PARP inhibitors described herein for the treatment of cancers such as AML, etc. method of hematological cancer). Additionally, pharmaceutical compositions comprising a CD33-targeted ADC containing an indolinyl-benzodiazepine dimer cytotoxic payload and a PARP inhibitor are also disclosed.

One embodiment of the invention is a method of treating cancer in a subject. In one embodiment, the cancer is selected from acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), B cell lineage acute lymphoblastic leukemia (B ALL), Chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), myelodysplastic syndrome (MDS), basal plasmacytoid DC neoplasia (BPDCN) leukemia, non-Hodgkin lymphomas (NHL) ), mantle cell lymphoma, and Hodgkin's leukemia (HL). In another embodiment, the cancer is chemotherapy-sensitive. In another embodiment, the cancer is chemotherapy resistant. In another embodiment, the cancer is acute myeloid leukemia (AML). In another embodiment, the AML is refractory or relapsed acute myeloid leukemia. In another embodiment, AML is characterized by overexpression of P-glycoprotein; overexpression of EVI1; changes in p53; mutations in DNMT3A; , BRCA2 or PALB2 mutations. The method comprises the steps of administering to the subject an effective amount of a PARP inhibitor and an effective amount of an ADC of formula (I):

or a pharmaceutically acceptable salt thereof. A double line between N and C represents a single or double bond, provided that when the double line is a double bond, X is absent and Y is hydrogen; and when the double line is a single bond, X is hydrogen , and Y is _-SO3H . The term "A" is an antibody or antigen-binding fragment that binds to CD33. Alternatively, "A" is an antibody or antigen-binding fragment that specifically binds to CD33 comprising the heavy chain variable region (VH) complementarity determining region (CDR) 1 sequence of SEQ ID NO:1, the VH of SEQ ID NO:2 The CDR2 sequence and the VH CDR3 sequence of SEQ ID NO:3, and the light chain variable region (VL) CDR1 sequence of SEQ ID NO:4, the VL CDR2 sequence of SEQ ID NO:5, and the VL CDR3 sequence of SEQ ID NO:6. The term "r" is an integer from 1 to 10.

In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7 or 9. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 8 or 10. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO:9 and a light chain variable region comprising the sequence of SEQ ID NO:10. In another embodiment, the antibody is huMy9-6. In another embodiment, the antibody is a CDR grafted or resurfaced antibody.

ADC1, ADC2, IMGN779 (defined below) and pharmaceutically acceptable salts thereof are specific examples of ADCs that can be used in the disclosed methods of treatment.

"A" is as defined for formula (I). The term "r" is an integer from 1 to 10. Methods of making ADC1, ADC2, and IMGN779 are provided in: US Pat. Nos. 8,765,740 and 9,353,127, the entire teachings of which are incorporated herein by reference.

Pharmaceutically acceptable salts are those suitable for use in humans and animals without undue toxicity, irritation and allergic reactions. Examples of suitable salts of ADCs of Formula (I) (ie, ADC1, ADC2, and IMGN779) are disclosed in US Pat. No. 8,765,740, the entire teachings of which are incorporated herein by reference. In one embodiment, the pharmaceutically acceptable salts of the ADCs of formula (I) (ie ADC1, ADC2 and IMGN779) are sodium or potassium salts.

Another embodiment of the present invention is a pharmaceutical composition comprising: i) an effective amount of a PARP inhibitor; ii) an effective amount of an antibody-drug conjugate of formula (I) (ie ADC1, ADC2, IMGN779) or a pharmaceutically acceptable salt; and iii) a pharmaceutically acceptable carrier or diluent. In one embodiment, the pharmaceutically acceptable salts of the ADCs of formula (I) (ie ADC1, ADC2 and IMGN779) are sodium or potassium salts.

Another embodiment of the present invention is an antibody-drug conjugate of formula (I) (ie ADC1, ADC2, IMGN779) or a pharmaceutically acceptable salt thereof for use in combination with a PARP inhibitor for the treatment of subjects with cancer tester. In one embodiment, the pharmaceutically acceptable salts of the ADCs of formula (I) (ie ADC 1, ADC 2 and IMGN779) are sodium or potassium salts. In one embodiment, the cancer is selected from acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), B cell lineage acute lymphoblastic leukemia (B ALL), Chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), myelodysplastic syndrome (MDS), basal plasmacytoid DC neoplasia (BPDCN) leukemia, non-Hodgkin lymphoma (NHL), mantle cell lymphoma tumor and Hodgkin's leukemia (HL). In another embodiment, the cancer is chemotherapy-sensitive. In another embodiment, the cancer is chemotherapy resistant. In another embodiment, the cancer is acute myeloid leukemia (AML). In another embodiment, the AML is refractory or relapsed acute myeloid leukemia. In another embodiment, AML is characterized by overexpression of P-glycoprotein; overexpression of EVI1; changes in p53; mutations in DNMT3A; , BRCA2 or PALB2 mutations.

Another embodiment of the invention is the use of an antibody-drug conjugate of formula (I) (ie ADC1, ADC2, IMGN779) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament in combination with a PARP inhibitor for the treatment of subjects with cancer. In one embodiment, the pharmaceutically acceptable salts of the ADCs of formula (I) (ie ADC1, ADC2 and IMGN779) are sodium or potassium salts. In another embodiment, the cancer is selected from acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), B cell lineage acute lymphoblastic leukemia (B ALL) , chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), myelodysplastic syndrome (MDS), basal plasmacytoid DC neoplasia (BPDCN) leukemia, non-Hodgkin's lymphoma (NHL), mantle cell Lymphoma and Hodgkin's Leukemia (HL). In another embodiment, the cancer is chemotherapy-sensitive. In another embodiment, the cancer is chemotherapy resistant. In another embodiment, the cancer is acute myeloid leukemia (AML). In another embodiment, the AML is refractory or relapsed acute myeloid leukemia. In another embodiment, AML is characterized by overexpression of P-glycoprotein; overexpression of EVI1; changes in p53; mutations in DNMT3A; , BRCA2 or PALB2 mutations.

definition

"IMGN779" means a CD33-targeted ADC comprising a huMy9-6 or Z4681A antibody conjugated to DGN462 via a cleavable disulfide linker (ie, comprising a heavy chain CDR1 having the sequence of SEQ ID NOs: 1-3, respectively) -3 and an antibody of light chain CDR1-3 having the sequence of SEQ ID NO:4-6; comprising a heavy chain variable region having the sequence of SEQ ID NO:9 and a light chain variable having the sequence of SEQ ID NO:10 region; or an antibody having a heavy chain sequence having the sequence of SEQ ID NO: 11 and an antibody having a light chain sequence having the sequence of SEQ ID NO: 12). The IMGN779 can be represented as ADC3 as shown below:

or a pharmaceutically acceptable salt thereof; or IMGN779 may also be represented as ADC4 as depicted below:

or a pharmaceutically acceptable salt thereof; or IMGN779 may be a combination of ADC3 and ADC4 or a pharmaceutically acceptable salt thereof.

"P-glycoprotein" means a polypeptide or fragment thereof that has at least about 85% amino acid sequence identity to the human sequence provided at NCBI Accession No. NP_001035830 and that confers multidrug resistance on cells in which it is expressed. The sequences of exemplary human P-glycoproteins are provided below:

"CD33 protein" means a polypeptide or fragment thereof having at least about 85% amino acid sequence identity to the human sequence provided at NCBI Accession No. CAD36509 and having anti-CD33 antibody binding activity. An exemplary human CD33 amino acid sequence is provided below:

"FLT3 protein", "FLT3 polypeptide", "FLT3", "FLT-3 receptor" or "FLT-3R" means the FLT3 tyrosine kinase receptor (also known as FLK- 2 and STK-1) human sequences having at least about 85%, 90%, 95%, 99% or 100% amino acid sequence identity and having tyrosine kinase activity (including receptor tyrosine kinase activity) polypeptides or its fragments. In one embodiment, the FLT3 amino acid sequence is the human FLT3 amino acid sequence provided below:

"FLT3-ITD" means a FLT3 polypeptide having internal tandem repeats (including but not limited to simple tandem repeats and/or tandem repeats with insertions). In various embodiments, the FLT3 polypeptide having internal tandem repeats is an activated FLT3 variant (eg, constitutively autophosphorylated). In some embodiments, the FLT3-ITD includes any exon or intron (including, for example, exon 11, exon 11 to intron 11, and exon 12, exon 14, exon 14 to tandem repeats in intron 14 and exon 15) and/or tandem repeats with insertions. Internal tandem repeat mutations (FLT3-ITD) are the most common FLT3 mutations present in approximately 20-25% of AML cases. Patients with FLT3-ITD AML had worse prognosis than those with wild-type (WT) FLT3, increased relapse rates, and shorter duration of response to chemotherapy.

"Analog" means a molecule that is not identical but has similar functional or structural characteristics. For example, a polypeptide analog retains the biological activity of the corresponding native polypeptide while having certain biochemical modifications that enhance the function of the analog relative to the native polypeptide. The biochemical modification can increase the protease resistance, membrane permeability or half-life of the analog without altering, for example, ligand binding. Analogs can include unnatural amino acids.

In this disclosure, "comprises" ("comprises", "comprising"), "containing" and "having" and the like may have the meanings ascribed to them under U.S. patent law, and may mean "includes," "includes," "including"), etc.; "consisting essentially of" or "consisting essentially of" also has the meaning ascribed to it under US Patent Law, and the terms are open ended to allow more than those stated, so long as The basic or novel features described are not modified by the presence of more than described, but do not include prior art embodiments.

"Substantially identical" means exhibiting at least 50% identity to a reference amino acid sequence (eg, any of the amino acid sequences described herein) or nucleic acid sequence (eg, any of the nucleic acid sequences described herein) of polypeptides or nucleic acid molecules. Preferably, the sequence is at least 60%, more preferably 80% or 85% and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (eg, the sequence analysis software package of the Genetic Computing Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or the PILEUP/PRETTYBOX program). The software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine acid; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, the BLAST program can be used, where a probability score between e ^-3 and e- ¹⁰⁰ indicates closely related sequences.

"Specifically binds" means an antibody or fragment thereof that recognizes and binds to a polypeptide of interest, but does not substantially recognize and bind to other molecules (which typically include a polypeptide of the invention) in a sample (eg, a natural sample).

A "subject" is a mammal, preferably a human, but can also be an animal in need of veterinary treatment, such as companion animals (eg, dogs, cats, etc.), farm animals (eg, cows, sheep, pigs, horses, etc.) etc.) and laboratory animals (eg, mice, mice, guinea pigs, etc.).

"Effective amount" means the amount of ADC or PARP inhibitor that elicits a desired biological response in a subject. The response includes alleviation of symptoms of the disease or disorder being treated, inhibition or delay of symptoms of the disease or recurrence of the disease itself, increased longevity of the subject compared to the absence of treatment, or inhibition or delay of symptoms of the disease or the disease itself. progress. Toxicity and therapeutic efficacy of ADC or PARP inhibitors can be determined by standard pharmaceutical procedures in cell cultures and experimental animals. The effective amount of the ADC or PARP inhibitor administered to a subject will depend on the stage, class and status of the multiple myeloma and the characteristics of the subject, such as general health, age, sex, weight, and drug tolerance. The effective amount of ADC or PARP inhibitor to be administered also depends on the route of administration and dosage form. Doses and intervals can be adjusted individually to provide plasma levels of the active compound sufficient to maintain the desired therapeutic effect.

The terms "treatment", "treat" and "treating" refer to reversing, moderating or inhibiting the progression of cancer as described herein or one or more symptoms thereof.

As used herein, the terms "administer", "administering", "administration" and the like refer to methods that can be used to deliver ADCs and PARP inhibitors to a desired site of biological action. These methods include, but are not limited to, intra-articular (in joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, subcutaneous, oral, topical, intrathecal, inhalation, transdermal, transrectal, etc. Wait. Techniques that can be used with the reagents and methods described herein can be found in, for example, Goodman and Gilman, The Pharmaceutical Theory of Basis of Therapeutics, current edition; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Company , Easton, Pa. In one aspect, the ADC and/or PARP inhibitor is administered intravenously.

As used herein, the term "or" should be understood to be inclusive unless explicitly stated or clear from context. As used herein, the terms "a" ("a", "an") and "the" should be construed in the singular or plural unless expressly stated otherwise or clear from the context.

Unless explicitly stated or clear from context, the term "about" as used herein is understood to mean within a range normally tolerated in the art, eg, within 2 standard deviations of the mean. About is understood to be at 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value Inside. Unless otherwise clear from the context, all numerical values provided herein are modified by the term about.

The listing of a listing of chemical groups in any definition of a variable herein includes the definition of the variable as any single group or combination of listed groups. The description of a variable or aspect herein in this embodiment includes the embodiment as any single embodiment or in combination with any other embodiment or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any other compositions and methods provided herein.

Description of drawings

Figure 1A shows HEL CD33+ AML cells treated with 500 pM IMGN779, 50 μM olaparib (Ola) or 500 pM IMGN779 + 50 μM olaparib and the effect on proliferation as measured by WST-8 reagent.

Figure IB shows the synergistic/additive effects of the IMGN779+olaparib combination in HEL cells calculated using Compusyn software. Data points below the line represent synergy between drug pairs.

Figure 2A shows MV4-11CD33+ AML cells treated with 750 pM IMGN779, 12 μM olaparib (Ola) or 750 pM IMGN779 + 12 μM olaparib and the effect on proliferation as measured by WST-8 reagent.

Figure 2B shows the synergistic/additive effect of the IMGN779+olaparib combination in MV4-11 cells calculated using Compusyn software. Data points below the line represent synergy between drug pairs.

Figure 3A shows HL60CD33+ AML cells treated with 25 pM IMGN779, 10 μM olaparib (Ola) or 25 μM IMGN779 + 10 μM olaparib and the effect on proliferation as measured by WST-8 reagent.

Figure 3B shows the synergistic/additive effect of the IMGN779+olaparib combination in HL60 cells calculated using Compusyn software. Data points below the line represent synergy between drug pairs.

Figure 4 shows cell viability and cell cycle effects in HEL cells treated with 500 pM IMGN779, 50 μM olaparib or 500 pm IMGN779 + 50 μM olaparib as assessed by flow cytometry.

Figure 5 shows the percentage of apoptosis in HEL cells treated with 500 pM IMGN779, 50 μM olaparib or 500 pm IMGN779 + 50 μM olaparib.

Figure 6A shows HEL cells treated with gradient concentrations of olaparib and the effect on cell death following DNA damaging radiation exposure at 0.5 Gy radiation.

Figure 6B shows HEL cells treated with graded concentrations of olaparib and the effect on cell death following DNA damaging radiation exposure at 0.75 Gy radiation.

Figure 7A shows the anti-leukemic activity of IMGN779 at different concentrations (30 μg/kg, 60 μg/kg and 100 μg/kg (by payload)) in a systemic HEL AML xenograft model.

Figure 7B shows the leukemia burden in mice at day 14 following administration of different concentrations (30 μg/kg, 60 μg/kg and 100 μg/kg (by payload)) of vehicle or IMGN779.

Figure 7C shows overall survival in a systemic HEL AML xenograft model treated with different concentrations of IMGN779 (30 μg/kg, 60 μg/kg and 100 μg/kg (by payload)).

Figure 8A shows the anti-leukemic activity of IMGN779 (15 μg/kg), olaparib (100 mg/kg) and IMGN779 (15 μg/kg) + olaparib (100 mg/kg) in a systemic HEL AML xenograft model.

Figure 8B shows that after administration of vehicle, IMGN779 alone (15 μg/kg), olaparib alone (100 mg/kg) or the combination of IMGN779 ((15 μg/kg) + olaparib (100 mg/kg) Leukemia burden in mice at day 22.

Figure 8C shows treatment with IMGN779 (15 μg/kg), olaparib (100 mg/kg) and IMGN779 ((15 μg/kg) + olaparib (100 mg/kg)) in a systemic HEL AML xenograft model overall survival rate.

Figure 9A shows the results of the CFU assay quantified 15 days after plating using a Spot-RT3 camera mounted on an inverted microscope with SPOT-Basic imaging software. A representative sample for each condition was captured, and triplicate wells were averaged and reported (+/- standard deviation).

Figure 9B shows the effect of 1 μM olaparib, 10 pM IMGN779 and olaparib (1 μM) + IMGN779 (10 pM) on cell colony formation in bone marrow samples from patients with relapsed/refractory AML. Cells were incubated at 37 °C for 15 days and then quantified using a Spot-RT3 camera mounted on an inverted microscope with SPOT-Basic imaging software. Representative sample images for each treatment condition are shown.

Figures 10A and 10B show treatment with various concentrations of rucaparib, velparib, niraparib, talazoparib and olaparib Post-proliferation of (A) HEL-luc and (B) HL60 cell lines.

Figures 11A, 11B and 11C show HEL-luc cells treated with: 800 pM IMGN779, 0.8 μM talazopanib (Tal), or 800 pM IMGN779 + 0.8 μM Tal ( FIG. 11A ); 800 pM IMGN779, 0.8 μM oligosaccharide Lapanib (Ola), 800 pM IMGN779 + 0.8 μM Ola (FIG. 11B); 800 pM IMGN779, 0.8 μM niraparib (Nir), or 800 pMIMGN779 + 0.8 μM Nir (FIG.11C), and measured by WST-8 reagent effect on proliferation.

Figures 12A and 12B show the synergistic/additive effects of the combination of IMGN779 + niraparib (Figure 12A) and the combination of IMGN779 + tarazopanib (Figure 12B) in HEL-luc cells calculated using Compusyn software. Data points below the line represent synergy between drug pairs.

Figures 13A, 13B, and 13C show percent apoptosis measured by flow cytometry in HEL-luc cells treated with: 800 pM IMGN779, 0.8 μM olaparib, or 800 pm IMGN779 + 0.8 μM olaparib (FIG. 13A); 800 pM IMGN779, 0.8 μM talaparib, or 800 pm IMGN779 + 0.8 μM talaparib (FIG. 13B); and 800 pM IMGN779, 0.8 μM niraparib, or 800 pm IMGN779 + 0.8 μM Nirapani (Figure 13C).

Figures 14A, 14B, and 14C show cell viability and cell cycle effects assessed by flow cytometry in HEL-luc cells treated with: 800 pM IMGN779, 0.8 μM talaparib, or 800 pm IMGN779 + 0.8 μM talaparib Laparib (FIG. 14A); 800 pM IMGN779, 0.8 μM olaparib, or 800 pm IMGN779 + 0.8 μM olaparib (FIG. 14B); and 800 pM IMGN779, 0.8 μM niraparib, or 800 pm IMGN779 + 0.8 μM Niraparib (Figure 14C).

Figures 15A, 15B, and 15C show the extent of DNA damage as measured by % positive for phosphorylated H2AX staining in HEL-luc cells treated with: 800 pm IMGN779, 0.8 μM talaparib, or 800 pm IMGN779 + 0.8 μM his Laparib (FIG. 15A); 800 pM IMGN779, 0.8 μM olaparib, or 800 pm IMGN779 + 0.8 μM olaparib (FIG. 15B); and 800 pM IMGN779, 0.8 μM niraparib, or 800 pm IMGN779 + 0.8 μM Niraparib (Figure 15C).

Detailed ways

The invention features the treatment of patients suffering from a disease by administering a CD33-targeted ADC containing an indolinyl-benzodiazepine dimer cytotoxic payload, in particular an ADC of formula (I) in combination with a PARP inhibitor Methods for patients with cancer, eg, hematological cancers such as AML.

The present invention is based, at least in part, on the discovery that IMGN779, a CD33-targeted antibody drug conjugate comprising an anti-huCD33 antibody conjugated to a novel DNA-alkylating agent DGN462 via a cleavable disulfide linker, also referred to as The combination of huMy9-6 or Z4681A) with olaparib was more active than the individual agents in vitro against primary patient AML cells and in vivo against AML xenografts in mice.

anti-CD33 antibody

In one embodiment, the antibody in the ADC (ADC1 or ADC2) of formula (I) is an anti-CD33 antibody, in particular a huMy9-6 antibody.

"My9-6", "murine My9-6" and "muMy9-6" are murine anti-CD33 antibodies from which huMy9-6 was derived. My9-6 was fully characterized with respect to germline amino acid sequences of light and heavy chain variable regions, amino acid sequences of light and heavy chain variable regions, identification of CDRs, identification of surface amino acids, and its expression in recombinant form. See, eg, US Patent Nos. 7,557,189; 7,342,110; 8,119,787; 8,337,855 and US Patent Publication No. 20120244171, each of which is incorporated herein by reference in its entirety. The amino acid sequence of muMy9-6 is also shown in Table 1 below. The My9-6 antibody was also functionally characterized and was shown to bind with high affinity to CD33 on the surface of CD33-positive cells.

The term "variable region" is used herein to describe certain portions of antibody heavy and light chains that differ in sequence in different antibodies and that cooperate with each other in the binding and specificity of each particular antibody for its antigen. Variability is generally unevenly distributed throughout the variable region of an antibody. It is usually concentrated in three segments of the variable region, called complementarity determining regions (CDRs) or hypervariable regions, both in the light and heavy chain variable regions. The more highly conserved parts of the variable regions are called framework regions. The variable regions of heavy and light chains comprise four framework regions largely adopting a beta-sheet configuration, each connected by three CDRs that form loops connecting the beta-sheet structure, and In some cases, part of a β-sheet structure is formed. The CDRs in each chain are held in close proximity by the framework regions, and CDRs from the other chain contribute to the formation of the antigen-binding site of the antibody (E.A. Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, 1991, NIH ). The "constant" regions are not directly involved in the binding of the antibody to the antigen, but instead exhibit various effector functions, such as the involvement of the antibody in antibody-dependent cellular cytotoxicity.

Table 1

Humanized versions of My9-6 have also been described, variously designated herein as "huMy9-6" and "humanized My9-6."

The goal of humanization is to reduce the immunogenicity of xenogeneic antibodies (eg, murine antibodies) for introduction into humans, while maintaining the antibody's full antigen-binding affinity and specificity. Humanized antibodies can be produced using several techniques such as resurfacing and CDR grafting. As used herein, resurfacing techniques use a combination of molecular modeling, statistical analysis, and mutagenesis to alter the non-CDR surfaces of antibody variable regions to resemble the surfaces of known antibodies of the target host.

Strategies and methods for resurfacing antibodies, as well as other methods for reducing the immunogenicity of antibodies in various hosts, are disclosed in US Pat. No. 5,639,641 (Pedersen et al.), which is incorporated herein by reference in its entirety. Briefly, in a preferred method, (1) a batch of antibody heavy and light chain variable regions is generated and aligned to give a set of surface-exposed positions of the heavy and light chain variable region frameworks, where all The aligned positions of the variable regions are at least about 98% identical; (2) define a set of heavy and light chain variable region framework surface exposed amino acid residues for rodent antibodies (or fragments thereof); (3) identify a group of rodent surface exposed amino acid residues most closely identical set of heavy chain and light chain variable region framework surface exposed amino acid residues; (4) heavy chain and light chain variable region framework surface exposed amino acid residues as defined in step (2); Chain variable region framework surface exposed amino acid residues are substituted with the set of heavy and light chain variable region framework surface exposed amino acid residues identified in step (3), except those determined by complementarity in rodent antibodies An amino acid residue within 5 Angstroms of any atom of any residue in the region; and (5) producing a humanized rodent antibody with binding specificity.

Antibodies can be humanized using various other techniques including CDR grafting (EP 0 239400; WO 91/09967; US Pat. Nos. 5,530,101; and 5,585,089), veneers or resurfacing (EP 0 592 106; EP 0 519596; Padlan E.A., 1991, Molecular Immunology 28(4/5):489-498; Studnicka G.M. et al., 1994, Protein Engineering 7(6):805-814; Roguska M.A. et al., 1994, PNAS 91:969-973) and chain shuffling (US Patent No. 5,565,332). Human antibodies can be prepared by a variety of methods known in the art, including phage display methods. See also US Patent Nos. 4,444,887, 4,716,111, 5,545,806 and 5,814,318; and International Patent Application Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741 (incorporated by reference in its entirety).

As described further herein, the CDRs of My9-6 were identified by modeling and their molecular structure was predicted. The My9-6 antibody was then prepared and fully characterized as described, eg, in US Pat. Nos. 7,342,110 and 7,557,189, which are incorporated herein by reference. The amino acid sequences of the light and heavy chains of a number of huMy9-6 antibodies are described, for example, in US Patent No. 8,337,855 and US Patent Publication No. 8,765,740, each of which is incorporated herein by reference. The amino acid sequences shown in Table 2 describe the huMy9-6 antibodies of the present invention.

Table 2

Although the murine My9-6 antibody and epitope-binding fragments of the humanized My9-6 antibody are discussed herein separately from the murine My9-6 antibody and its humanized versions, it should be understood that the term "antibody" or "antibody" of the invention "Antibody" may include full-length muMy9-6 and huMy9-6 antibodies as well as epitope-binding fragments of these antibodies.

In another embodiment, an antibody or epitope-binding fragment thereof is provided comprising at least one complementarity determining region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and having the ability to bind CD33.

In another embodiment, there is provided an antibody or epitope-binding fragment thereof comprising at least one heavy chain variable region and at least one light chain variable region, wherein the heavy chain variable region comprises three variable regions having three variables each represented by SEQ ID NO. : the complementarity determining regions of the amino acid sequences represented by 1-3, and wherein the light chain variable region comprises three complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 4-6, respectively.

In another embodiment, there is provided an antibody having a heavy chain variable region that shares at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 7, more preferably with SEQ ID NO: 7 Amino acids that share 95% sequence identity, most preferably 100% sequence identity to SEQ ID NO:7.

Similarly, antibodies are provided having a light chain variable region that shares at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:8, more preferably shares 95% with SEQ ID NO:8 % sequence identity, most preferably amino acids that share 100% sequence identity with SEQ ID NO:8.

In another embodiment, antibodies are provided having humanized (eg, resurfaced, CDR-grafted) heavy chain variable regions that share the amino acid sequence represented by SEQ ID NO:9 At least 90% sequence identity, more preferably 95% sequence identity with SEQ ID NO:9, most preferably 100% sequence identity with SEQ ID NO:9.

Similarly, antibodies are provided having humanized (eg, resurfaced, CDR-grafted) light chain variable regions that share at least 90% sequence with the amino acid sequence corresponding to SEQ ID NO: 10 The identity, more preferably shares 95% sequence identity with SEQ ID NO:10, most preferably shares 100% sequence identity with SEQ ID NO:10. In certain embodiments, the antibody includes conservative mutations in framework regions outside the CDRs.

As used herein, an "antibody fragment" includes any portion of an antibody that retains the ability to bind CD33, commonly referred to as an "epitope-binding fragment." Examples of antibody fragments preferably include, but are not limited to, Fab, Fab' and F(ab') ₂ , Fd, single-chain Fv (scFv), single-chain antibody, disulfide-linked Fv (sdFv) and _VL or Fragments of _VH domains. Epitope binding fragments (including single chain antibodies) may comprise variable regions alone or in combination with all or part of the following: hinge region, _CH1 , _CH2 and _CH3 domains. The fragments may contain one or both of Fab fragments or F(ab') ₂ fragments. Preferably, antibody fragments contain all six CDRs of the entire antibody, but fragments containing less than all of these regions (eg, three, four or five CDRs) are also functional. Furthermore, functional equivalents may be or may be combined members of any of the following immunoglobulin classes: IgG, IgM, IgA, IgD or IgE and subclasses thereof. Fab and F(ab') ₂ fragments can be produced by proteolytic cleavage using enzymes such as papain (Fab fragments) or pepsin (F(ab') ₂ fragments). A single-chain FV (scFv) fragment is an epitope-binding fragment comprising at least one fragment of an antibody heavy chain variable region ( _VH ) linked to at least one fragment of an antibody light chain variable region ( _VL ). The linker can be a short, flexible peptide selected to ensure that correct three-dimensional folding of the ( _VL ) and ( _VH ) regions occurs once they are ligated, thereby maintaining the target molecule binding specificity of the entire antibody. Single chain antibody fragments are derived from the whole antibody. The carboxy terminus of the ( _VL ) or ( _VH ) sequence can be covalently linked to the amino acid terminus of the complementary ( _VL ) and ( _VH ) sequences via a linker. Single chain antibody fragments can be produced by molecular cloning, antibody phage display libraries, or similar techniques well known to those of skill in the art. These proteins can be produced, for example, in eukaryotic cells or prokaryotic cells, including bacteria.

Epitope-binding fragments of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying polynucleotide sequences encoding functional antibody domains. In particular, such phage can be used to display epitope binding domains expressed from a series or combinatorial antibody library (eg, human or murine). Phage expressing an epitope-binding domain that binds an antigen of interest can be selected or identified with an antigen, eg, using labeled CD33 or CD33 bound or captured to a solid surface or beads. Phage used in these methods are typically filamentous phage comprising fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains reconstituted Fusion to phage gene III or gene VIII protein.

Examples of phage display methods that can be used to prepare the epitope-binding fragments of the invention include those disclosed in: Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCT Publication Nos. PCT/GB91/01134; PCT Publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;

Following selection of phage, regions encoding fragmented phage can be isolated and used, for example, by recombinant DNA techniques as described in detail below, by expression in selected hosts, including mammalian cells, insect cells, plant cells, yeast, and bacteria. Generation of epitope-binding fragments. For example, techniques for the recombinant production of Fab, Fab' and F(ab') ₂ fragments can also be employed using methods known in the art, such as those disclosed in: PCT Publication WO 92/22324 Mullinax et al., 1992, BioTechniques 12(6): 864-869; Sawai et al., 1995, AJRI 34: 26-34; and Better et al., 1988, Science 240: 1041-1043; enter. Examples of techniques that can be used to generate single-chain Fvs and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology 203:46-88; Shu et al., 1993, PNAS 90: 7995-7999; Skerra et al., 1988, Science 240:1038-1040.

Also included within the scope of the invention are the My9-6 antibody and functional equivalents of the humanized My9-6 antibody. The term "functional equivalents" includes antibodies, chimeric antibodies, modified antibodies, and artificial antibodies having homologous sequences, eg, where each functional equivalent is defined by its ability to bind CD33. Those skilled in the art will understand that there is overlap between the group of molecules referred to as "antibody fragments" and the group referred to as "functional equivalents."

Antibodies having homologous sequences are those having amino acid sequences that have sequence identity or homology to the amino acid sequences of the murine My9-6 and humanized My9-6 antibodies of the invention. Preferably, there is identity to the amino acid sequence of the variable regions of the murine My9-6 and humanized My9-6 antibodies of the invention. "Sequence identity" and "sequence homology" as applied to amino acid sequences herein are defined as, for example, as determined by the FASTA search method according to Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988) The sequence is determined to have at least about 90%, 91%, 92%, 93% or 94% sequence identity to another amino acid sequence, and more preferably at least about 95%, 96%, 97%, 98% or 99% sequence identity sex.

As used herein, a chimeric antibody is one in which different portions of the antibody are derived from different animal species. For example, antibodies having variable regions derived from murine monoclonal antibodies paired with human immunoglobulin constant regions. Methods for producing chimeric antibodies are known in the art. See, eg, Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; US Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which The entire contents are incorporated herein by reference.

The CDRs are most important for epitope recognition and antibody binding. However, CDR-containing residues can be altered without interfering with the ability of the antibody to recognize and bind its cognate epitope. For example, changes can be made that do not affect epitope recognition, but that increase the binding affinity of the antibody for the epitope.

Accordingly, also included within the scope of the present invention are improved forms of murine and humanized antibodies that also specifically recognize and bind CD33, preferably with increased affinity.

Based on knowledge of the primary antibody sequence and its properties (eg, binding and expression levels), several studies have investigated the effects of introducing one or more amino acid changes at different positions in the antibody sequence (Yang, W.P. et al., 1995, J. Mol. Biol ., 254, 392-403; Rader, C. et al., 1998, Proc. Natl. Acad. Sci. USA, 95, 8910-8915; Vaughan, T. J. et al., 1998, Nature Biotechnology, 16, 535-539).

In these studies, the heavy chains in the CDR1, CDR2, CDR3 or framework regions were altered by using methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, or mutator strains of E. coli and light chain gene sequences, yielding equivalents of primary antibodies (Vaughan, T.J. et al., 1998, Nature Biotechnology, 16, 535-539; Adey, N.B. et al., 1996, Chapter 16, pp. 277-291, "Phage Display of Peptides and Proteins", Kay, B.K. et al. eds. Academic Press). These methods of altering the sequence of primary antibodies have improved the affinity of secondary antibodies (Gram, H. et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 3576-3580; Boder, E.T. et al., 2000, Proc. . Natl. Acad. Sci. USA, 97, 10701-10705; Davies, J. and Riechmann, L., 1996, Immunotechnolgy, 2, 169-179; Thompson, J. et al., 1996, J. Mol. Biol., 256, 77-88; Short, M.K. et al., 2002, J. Biol. Chem., 277, 16365-16370; Furukawa, K. et al., 2001, J. Biol. Chem., 276, 27622-27628).

The antibody sequences described herein (eg, in Tables 1 and 2) can be used to develop anti-CD33 antibodies with improved function, including improved CD33 affinity, through a similar targeting strategy of altering one or more amino acid residues of an antibody. Improved antibodies also include those with improved characteristics, prepared by standard techniques of animal immunization, hybridoma formation, and selection of antibodies with specific characteristics.

CD33-targeted antibody drug conjugates

In certain embodiments, the present invention provides methods of treating cancer (eg, hematological cancer) in a subject comprising administering to the subject an effective amount of olaparib, or a pharmaceutically acceptable salt thereof, and an effective amount The ADC of formula (I):

or a pharmaceutically acceptable salt thereof. Double line between N and C represents a single or double bond, provided that when the doublet is a double bond, X is absent and Y is hydrogen; and when the doublet is a single bond, X is hydrogen and Y is _-SO3H . The term "A" is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising the heavy chain variable region (VH) complementarity determining region (CDR) 1 sequence of SEQ ID NO:1, the VH of SEQ ID NO:2 The CDR2 sequence and the VH CDR3 sequence of SEQ ID NO:3, and the light chain variable region (VL) CDR1 sequence of SEQ ID NO:4, the VL CDR2 sequence of SEQ ID NO:5, and the VLCDR3 sequence of SEQ ID NO:6. The term "r" is an integer from 1 to 10.

In one embodiment, the antibody or antigen-binding fragment thereof includes a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7 or 9. In another embodiment, the antibody or antigen-binding fragment thereof includes a light chain variable region comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 8 or 10. In one embodiment, the antibody is huMy9-6. In another embodiment, the antibody is a CDR grafted or resurfaced antibody.

ADC1, ADC2, IMGN779, and pharmaceutically acceptable salts thereof are specific examples of ADCs that can be used in the disclosed methods of treatment.

"A" is as defined for formula (I). The term "r" is an integer from 1 to 10. Methods of making ADC1, ADC2, and IMGN779 are provided in: US Pat. Nos. 8,765,740 and 9,353,127, the entire teachings of which are incorporated herein by reference.

In other embodiments, the antibody portion of the ADC of Formula (I) (ie, ADC1 or ADC2) is an anti-CD33 antibody comprising at least about 90%, 91%, 92%, 93%, or 94% with SEQ ID NO:9 % sequence identity and more preferably at least about 95%, 96%, 97%, 98% or 99% sequence identity heavy chain variable region and at least about 90%, 91%, 92% with SEQ ID NO: 10 , 93% or 94% sequence identity, and more preferably light chain variable regions of at least about 95%, 96%, 97%, 98% or 99% sequence identity.

In particular embodiments, the antibody portion of the ADC of Formula (I) (ie, ADC1 or ADC2) is the huMy9-6 antibody, also referred to as "Z4681A." In a specific embodiment, the CD33-targeted ADC is IMGN779. IMGN779 includes huMy9-6 or Z4681A antibodies conjugated to DGN462 via a cleavable disulfide linker. The IMGN779 can be represented as ADC3 as shown below:

or a pharmaceutically acceptable salt thereof; or IMGN779 may also be represented hereinafter as ADC4:

or a pharmaceutically acceptable salt thereof; or the IMGN can be a combination of ADC3 and ADC4.

In certain embodiments, the conjugates described herein can include 1-10 cytotoxic benzodiazepine dimer compounds, 2-9 cytotoxic benzodiazepine dimer compounds, 3- 8 cytotoxic benzodiazepine dimer compounds, 4-7 cytotoxic benzodiazepine dimer compounds or 5-6 cytotoxic benzodiazepine dimer compounds.

In certain embodiments, compositions comprising the conjugates described herein may comprise an average of 1-10 cytotoxic benzodiazepine dimer molecules per antibody molecule. The average ratio of cytotoxic benzodiazepine dimer molecules per antibody molecule is referred to herein as the drug-to-antibody ratio (DAR). In one embodiment, the DAR is between 2-8, 3-7, 3-5, or 2.5-3.5.

The cytotoxic benzodiazepine dimer compounds and conjugates described herein can be prepared according to the methods described in US Pat. Nos. 8,765,740 and 9,353,127, such as, but not limited to, paragraphs [0395]-[0397] ] and [0598]-[0607], Figures 1, 15, 22, 23, 38-41, 43, 48, 55, and 60, and Examples 1, 6, 12, 13, 20, 21, 22, 23, 26-30 and 32 and paragraphs [0007]-paragraphs [0105], paragraphs [0197]-paragraphs [0291], Figures 1-11, 16, 28, and Examples 1-7, 9-13, 15 and 16 of US Patent No. 9,353,127.

The term "cationic" refers to a positively charged ion. Cations can be monovalent (eg, Na ⁺ , K ⁺ , etc.), divalent (eg, Ca2 ⁺ , ^Mg2+ , etc.) or polyvalent (eg, Al3 ⁺ , etc.). Preferably, the cation is monovalent.

The phrase "pharmaceutically acceptable" indicates that a substance or composition must be chemically and/or toxicologically compatible with the other ingredients of the formulation comprising and/or the mammal being treated therewith.

The phrase "pharmaceutically acceptable salt" as used herein refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the present invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isoflurane acid salt, lactate, salicylate, acid citrate, tartrate, oleate, tannin, pantothenate, hydrogen tartrate, ascorbate, succinate, maleate, Gentianate, Fumarate, Gluconate, Glucuronate, Sugar Salt, Formate, Benzoate, Glutamate, Methanesulfonate "Mesylate", Ethane Sulfonates, benzenesulfonates, p-toluenesulfonates, pamoate (ie, 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)), alkali metals (eg, sodium and potassium) salts, alkaline earth metal (eg, magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may include the inclusion of another molecule, such as acetate ion, succinate ion, or other counter ion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Additionally, pharmaceutically acceptable salts may have more than one charged atom in their structure. Instances where multiple charged atoms are part of a pharmaceutically acceptable salt may have multiple opposing ions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counter ions. In specific embodiments, the pharmaceutically acceptable salt is a sodium or potassium salt.

If the compound of the present invention is a base, the desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art, for example, with a mineral acid (eg, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, etc.) or with organic acids (such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranoside (such as glucuronic acid) or galacturonic acid), alpha hydroxy acids (such as citric acid or tartaric acid), amino acids (such as aspartic acid or glutamic acid), aromatic acids (such as benzoic acid or cinnamic acid), sulfonic acids (such as p-toluenesulfonic acid) acid or ethanesulfonic acid) or the like) to treat the free base.

If the compound of the present invention is an acid, the desired pharmaceutically acceptable salt can be prepared by any suitable method, for example, with an inorganic or organic base (eg, an amine (primary, secondary or tertiary), an alkali metal hydrogen oxides or alkaline earth metal hydroxides or the like) to treat the free acid. Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids (eg, glycine and arginine), ammonia, primary, secondary, and tertiary amines, and cyclic amines (eg, piperidine, morpholine) and piperazine), and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

PARP inhibitors

PARP refers to a family of poly-ADP ribose polymerases that are involved in a variety of DNA-related functions, including cell proliferation, differentiation, apoptosis, DNA repair, and also have effects on telomere length and chromosomal stability (d'Adda di Fagagna et al., 1999, Nature Gen., 23(1):76-80). "PARP inhibitor" refers to a substance that selectively binds to and reduces the activity of poly-ADP ribose polymerase. In one aspect, the PARP inhibitor used in the disclosed methods inhibits PARP-1 or PARP-2. PARP-1 is a poly-ADP ribose polymerase encoded by the PARP-1 gene. (See NCBI, 2016, PARP1 poly(ADP-ribose) polymerase 1, [Homo sapiens (human)]. PARP-2 is a poly-ADP ribose polymerase encoded by the PARP-1 gene. (See NCBI, 2016, PARP2poly(ADP) -ribose) polymerase 2, [Homo sapiens (human)].PARP inhibitors can be identified using methods known in the art. See, for example, Cheung etc., "Ascintillation proximity assay for poly(ADP-ribose) polymerase," Anal. Biochem. 2000, Vol. 282, pp. 24-28.

Suitable PARP inhibitors include those designed as analogs of benzamides that compete with the natural substrate NAD ⁺ for binding in the catalytic site of PARP. These PARP inhibitors include, but are not limited to, benzamides, quinolinones and isoquinolinones, benzopyrones, 3,5-diiodo-4-(4'-methoxy-3',5 Methyl '-diiodo-phenoxy)benzoate (US 5,464,871, US 5,670,518, US 5,922,775, US 6,017,958, US 5,736,576 and US 5,484,951, each of which is incorporated herein by reference in its entirety). Other suitable PARP inhibitors include various cyclic benzamide analogs (ie, lactams), which are potent inhibitors at the NAD ⁺ site. Other PARP inhibitors include, but are not limited to, benzimidazoles and indoles (see, eg, EP 841924, EP 127052, US 6,100,283, US 6,310,082, US 2002/156050, US 2005/054631, WO 05/012305, WO 99/ 11628 and US2002/028815, each of which is hereby incorporated by reference in its entirety).

PARP inhibitors can have the following structural features: 1) an amide or lactam functional group; 2) the NH proton of the amide or lactam functional group can be reserved for efficient bonding; 3) an amide group attached to an aromatic ring or with Aromatic ring-fused lactam groups; 4) optimal cis conformation of amides in the aromatic plane; and 5) confinement of mono-arylcarboxamides to heteropolycyclic lactams (Costantino et al., 2001, J Med Chem., 44:3786-3794); Virag et al., 2002, Pharmacol Rev., 54:375-29, which outline various PARP inhibitors and are each incorporated herein by reference in their entirety. Some examples of PARP inhibitors include, but are not limited to, isoquinolinones and dihydroisoquinolinones (eg, US 6,664,269 and WO 99/11624, each of which is hereby incorporated by reference in its entirety), nicotinamide , 3-aminobenzamide, monoaryl acyl and bicyclic, tricyclic or tetracyclic lactams, phenidinone (Perkins et al., 2001, Cancer Res., 61:4175-4183, which is incorporated by reference in its entirety) incorporated herein), 3,4-dihydro-5-methyl-isoquinolin-1(2H)-one, and benzoxazole-4-carboxamide (Griffin et al., 1995, Anticancer Drug Des, 10:507 -514; Griffin et al., 1998, J Med Chem, 41:5247-5256; and Griffin et al., 1996, Pharm Sci, 2:43-48, each of which is hereby incorporated by reference in its entirety), dihydroisoquinoline Lin-1(2H)-one, 1,6-naphthyridin-5(6H)-one, quinazolin-4(3H)-one, thieno[3,4-c]pyridin-4(5H)-one and thieno[3,4-d]pyrimidin-4(3H)-one, 1,5-dihydroxyisoquinoline and 2-methyl-quinazolin-4[3H]-one (Yoshida et al., 1991, J Antibiot (Tokyo, ) 44: 111-112; Watson et al., 1998, Bioorg MedChem., 6:721-734; and White et al., 2000, J Med Chem., 43:4084-4097, each of which is incorporated by reference in its entirety manner incorporated herein), 1,8-naphthalimide (Banasik et al., 1992, J Biol Chem, 267:1569-1575; Watson et al., 1998, Bioo 2001, Nat Med., 7:108-113; Li et al., 2001, Bioorg Med Chem Lett., 11:1687-1690 30:1071-1082, each of which is hereby incorporated by reference in its entirety), tetracyclic lactam, 1,11b-dihydro-[1] Benzopyrano-[4,3,2-de]isoquinolin-3[2H]-one, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Zhang et al., 2000, Biochem Biophys Res Commun., 278:590-598; and Mazzon et al., 2001, Eur J Pharmacol, 415:85-94, each of which is incorporated herein by reference in its entirety). Other examples of PARP inhibitors include, but are not limited to, those PARP inhibitors detailed in the following patents: US 5,719,151, US 5,756,510, US 6,015,827, US 6,100,283, 6,201,020、US 6,235,748、6,306,889、US 6,346,536、US 6,380,193、US 6,387,902、US 6,395,749、US 6,426,415、US 6,514,983、US 6,723,733、US 6,448,271、US 6,495,541、US 6,548,494、US 6,500,823、US 6,664,269、US 6,677,333、US 6,903,098、US6 , 924,284, US 6,989,388, US 6,277,990, US 6,476,048, and US 6,531,464, each of which is incorporated herein by reference in its entirety. PARP抑制剂的额外实例包括(但不限于)以下专利申请公开案中详述的那些PARP抑制剂：US 2004198693Al、US 2004034078A1、US2004248879A1、US2004249841A 2005080096A1、US 2005171101Al、US2005054631A1、WO 05054201A1、WO05054209A1、WO 05054210Al、 WO05058843A1, WO 06003146A1, WO 06003147A1, WO06003148A1, WO06003150A1 and WO 05097750A1, each of which is incorporated herein by reference in its entirety.

The following are specific examples of PARP inhibitors that can be used in the disclosed invention:

(Olapani); (Taprazopanib);

(Nirapani); (Vilipani);

(Rukapani);

ABT767; and MP-124

or a pharmaceutically acceptable salt thereof. In a specific embodiment, the present invention provides a method of treating cancer, wherein the PARP inhibitor is olaparib or a pharmaceutically acceptable salt thereof. In another specific embodiment, the present invention provides a method of treating cancer, wherein the PARP inhibitor is taprazopanib or a pharmaceutically acceptable salt thereof.

therapeutic application

The present invention provides methods of treating patients suffering from cancer, particularly hematological cancers such as AML, by administering a combination of a CD33-targeted ADC and a PARP inhibitor. As used herein, a "hematological cancer" is a cancer that begins in blood-forming tissues (eg, bone marrow) or in cells of the immune system. Examples of hematological cancers are leukemia, lymphoma and multiple myeloma.

Cancers that can be treated using the disclosed methods include leukemia, lymphoma, and myeloma. The cancer may be chemotherapy-sensitive; alternatively, the cancer may be chemotherapy-resistant. More specifically, cancers that can be treated using the disclosed methods include lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute myeloid leukemia Cellular leukemia (APL), myelodysplastic syndrome (MDS), acute monocytic leukemia (AMOL), hairy cell leukemia (HCL), T-cell prelymphocytic leukemia (T-PLL), large granular lymphoid T-cell leukemia, adult T-cell leukemia, small lymphocytic lymphoma (SLL), Hodgkin lymphoma (nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte-depleted or not, and nodular lymphocytic predominant Hodgkin lymphoma), non-Hodgkin lymphoma (all subtypes), chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prelymphocytic leukemia, lymphoplasmacytic lymphoma neoplasms (eg, Waldenstrom's macroglobulinemia ( macroglobulinemia)), splenic marginal zone lymphoma, plasma cell neoplasia (plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease), extranodal marginal zone B-cell lymphoma (MALT lymphoma) , nodular marginal zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma , primary effusion lymphoma, Burkitt lymphoma/leukemia, T-cell prelymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell leukemia/ Lymphoma, extranodal NK/T-cell lymphoma (nasal), enteropathy T-cell lymphoma, hepatosplenic T-cell lymphoma, NK blast lymphoma, mycosis fungoides/Sezari syndrome ( sezarysyndrome), primary cutaneous CD30-positive T-cell lymphoproliferative disorder, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma (nonspecific finger), degenerative large cell lymphoma), and multiple myeloma (plasma cell myeloma Kahler's disease).

In another embodiment, the cancer is selected from acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), B cell lineage acute lymphoblastic leukemia (B ALL) , chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), myelodysplastic syndrome, basal plasmacytoid DC neoplasia (BPDCN) leukemia, non-Hodgkin lymphoma (NHL), mantle cell lymphoma and Hodgkin's Leukemia (HL). In another embodiment, the cancer is acute myeloid leukemia (AML). In another embodiment, the acute myeloid leukemia is refractory or relapsed acute myeloid leukemia. In other embodiments, the present invention provides treatment of patients with multi-drug resistant AML. P-glycoprotein (PGP) (also known as MDR1) is a 170 kD ATP-dependent drug efflux pump. It is a member of the ABC superfamily and is abundantly expressed in multidrug resistant (MDR) cells and produced by the ABCB1 gene. AML cells expressing PGP are at least partly resistant to treatment with conventional chemotherapeutic agents. Accordingly, the present invention also provides methods of treating PGP-expressing AML.

The present invention also provides methods of treating hematological cancers with at least one negative prognostic factor (eg, overexpression of P-glycoprotein, overexpression of EVI1, p53 changes, DNMT3A mutations, FLT3 internal tandem repeats, and/or complex karyotypes) . In other embodiments, the present invention also provides methods of treating hematological cancers with reduced expression in BRCA1, BRCA2 or PALB2 or with mutations in BRCA1, BRCA2 or PALB2. It is also within the scope of the invention to select patients with at least one negative prognostic factor and/or reduced expression or mutation of BRCA1, BRCA2 or PALB2 prior to administration of the combination of a CD-33 targeted ADC and a PARP inhibitor.

In certain embodiments, the CD33-targeted ADC is administered to a subject in a pharmaceutically acceptable dosage form. ADCs can be administered intravenously by bolus injection or by continuous infusion over a period of time, by intramuscular, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes. Pharmaceutical compositions containing ADCs are administered by intratumoral, peritumoral, intralesional or perilesional routes to exert local and systemic therapeutic effects.

A pharmaceutically acceptable dosage form will generally include pharmaceutically acceptable agents such as carriers, diluents and excipients. These agents are well known, and one skilled in the art can determine the most appropriate agent based on the clinical situation. Examples of suitable carriers, diluents and/or excipients include: (1) Dubeck's Phosphate Buffered Saline, pH about 7.4, containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose.

When presented in an aqueous dosage form rather than lyophilized, a CD33-targeted ADC will typically be formulated at a concentration of about 0.1 mg/ml to 100 mg/ml, although wide variations outside these ranges are permitted. For the treatment of disease, the appropriate dose of the CD33-targeted ADC will depend on the type of disease to be treated, the severity and course of the disease, the course of previous treatments, the patient's medical history and response to antibodies, and the attending physician, as defined above free decision. The antibody is suitably administered to the patient at one time or in a series of treatments.

In the disclosed method, the ADC and the PARP inhibitor are administered in combination. Combination therapy is meant to include administration of two or more therapeutic agents to a single subject, and is intended to include treatment regimens in which the agents are administered by the same or different routes of administration or at the same or different times. These terms include administering two or more agents to a subject such that both agents and/or their metabolites are present in the subject at the same time. This includes simultaneous administration in separate compositions, simultaneous administration in the same composition and administration at different times in different compositions.

ADCs for use in the disclosed methods and pharmaceutical compositions can be supplied as solutions or lyophilized powders that are tested for sterility and endotoxin levels. Suitable pharmaceutically acceptable carriers, diluents and excipients are well known and can be determined by those skilled in the art based on the clinical situation.

Examples of suitable carriers, diluents and/or excipients include: (1) Dubeck's Phosphate Buffered Saline, pH about 7.4, with or without about 1 mg/ml to 25 mg/ml of human serum albumin , (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose; and may also contain antioxidants such as tryptamine and stabilizers such as Tween 20.

Disclosed are pharmaceutical compositions comprising an ADC, a PARP inhibitor, and generally at least one additional substance (eg, a pharmaceutically acceptable carrier or diluent). The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. In embodiments, the compositions are formulated according to conventional procedures as pharmaceutical compositions suitable for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to human organisms.

The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assays, screening, and therapeutic methods of the present invention, and are not intended to limit the scope of the invention that the inventors regard as their invention.

Example

Example 1. Combination of IMGN779 with olaparib shows enhanced anti-leukemia activity, reduces cell survival, induces S-phase arrest, and increases apoptosis in vitro

Human CD33+ AML cell lines (HEL, MV4-11 and HL60) were treated in vivo with control, IMGN779, olaparib or IMGN779 and olaparib. Proliferation was measured by WST-8 reagent. Synergistic/additive effects were calculated using Compusyn software. Flow cytometry was performed to evaluate apoptosis, cell viability and cell cycle effects.

IMGN779 treatment induced significant in vitro growth inhibition in all tested CD33+ human AML cell lines in a dose-dependent manner. The human AML cell lines tested highly expressed human CD33 and IMGN779 cell killing was observed to be CD33-dependent. Olaparib also exerted dose-dependent in vitro growth inhibition in all CD33+ human AML cell lines tested and induced cell death in human AML cell lines through reversal of DNA damage repair mechanisms (data not shown). Combination treatment with IMGN779 (25 pM-750 pM) and olaparib (10-50 μM) significantly enhanced the anti-leukemic effect in monotherapy in the same cell lines (FIG. 1A, FIG. 2A and FIG. 3A). The combination index of IMGN779 and olaparib treatment ranged from 0.7 to 0.9, consistent with a synergistic effect (Figure 1B, Figure 2B and Figure 3B). The combination of IMGN779 with olaparib significantly reduced overall cell survival, increased apoptosis, and induced nearly complete S-phase cell cycle arrest compared to control and single-agent treatments (Figures 4 and 5) . The effect on cell death following DNA damaging radiation exposure in the presence of olaparib alone was also assessed, showing that increases in cell death and apoptosis were associated with increased olaparib concentrations, supporting a mechanism of action of PARP inhibitors (FIGS. 6A and 6B).

Example 2. Combination of IMGN779 with olaparib reduces AML burden and prolongs survival in a systemic AML xenograft model

In these experiments, stably transfected luciferase-positive human AML cells (HEL) characterized by high hCD33 expression levels were injected via the tail vein into 6-8 week old SCID mice that were not yet lethally irradiated. Leukemic transplanted animals were divided into treatment groups consisting of vehicle control, IMGN779 (15mcg/kg), olaparib (100 mg/kg) or IMGN779 + olaparib (same dose). In vivo systemic leukemia disease burden was assessed weekly by small animal bioluminescence imaging. Toxicity was determined by clinical examination and body weight measurements throughout the experiment. The overall study endpoint was (a) time to overall morbidity/mortality and/or (b) change in leukemia disease burden compared to vehicle-treated mice or mice treated with single-agent therapy. 8-10 mice were used in the control and experimental groups as it has been found that this is the minimum number of animals required to determine statistical significance using the Cox-Mandel test. Simple statistical analysis will be performed using Stat Prism statistical software.

IMGN779 administered by payload (0.5 mg/kg to 5 mg/kg, via antibody) in single doses ranging from 30 μg/kg to 100 μg/kg carried systemic human CD33+ AML (HEL-luciferase) in SCID mice ) xenografts were generally well tolerated in SCID mice. Significant dose-dependent antileukemic activity as reflected by reduced leukemia burden and prolonged overall survival was observed (FIG. 7A-FIG. 7C). As shown in Figures 8A-8C, vehicle treatment (median 35.3 days, p 0.0189), IMGN779 alone (median 40.2 days, p 0.0283) or olaparib alone (median 33.7 days, p0.0009), mice engrafted with human AML cells (HEL-luciferase) and treated with the combination of IMGN779 and olaparib had significantly longer overall survival compared to vehicle (median 46.2 days from vaccination ). On day 22 of treatment, overall leukemia disease burden, as determined by systemic bioluminescence flux in mice in the combination treatment group, was significantly reduced as opposed to vehicle or monotherapy. The results of these studies demonstrate that the combination of IMGN779 with olaparib enhances antitumor activity in HEL-luciferase systemic AML xenografts.

Example 3. Combination of IMGN779 with olaparib enhances the inhibition of primary AML colony formation

Assess IMGN779 in patients with known CD33 expression levels, disease status (relapsed vs. secondary vs. refractory/relapsed), karyotype, molecular aberrations (i.e., FLT-3 and NPM-1 mutational status), and response to therapy In vitro efficacy in up to 50 clinically annotated patient AML samples, if any.

A short-term colony forming unit (CFU) assay was performed using cells obtained from multiple patients to assess the preclinical efficacy of IMGN779 in primary AML samples. Cryopreserved AML patient samples were obtained under an IRB-approved protocol from Roswell Park Hematologic Procurement SharedResource. Thaw cells on the day of the assay. Quantification of surface expression of the human CD33 molecule was performed on the same day on patient AML samples using Quantibright bead assays. Thawed cells were assessed for overall viability; samples with >50% viable cells were further quantified and exposed in vitro to vehicle (PBS) or varying concentrations of IMGN779 and/or olapanida for 24 hours, after Inoculate in semi-solid methylcellulose medium for 13-15 days. CFU assays were quantified 13–15 days after methylcellulose culture inoculation using a Spot-RT3 camera mounted on an inverted microscope with SPOT-Basic imaging software. A representative sample for each condition was captured, and triplicate wells were averaged and reported (+/- standard deviation), as seen in Figure 9A.

CFU assays were established for primary AML samples treated with vehicle and various concentrations of single agent IMGN779 CFU. Information on clinical features (particularly diagnostic cytogenetics and FLT-3 mutation status) is provided and is supplied by the RPCI Hematologic Procurement Shared Resource Facility under an IRB-approved protocol. IMGN779 was found to inhibit primary AML sample colony formation in a dose-dependent manner in a total of 15 primary AML samples. IMGN779 was combined with olaparib to verify the synergistic nature of the combination in primary AML samples. Combination doses were performed in triplicate. Representative sample images for each treatment condition are shown in Figure 9B. Significance between treatment groups was determined using an unpaired t-test. Exposure to IMGN779 in combination with olaparib significantly inhibited CFU growth of progenitor cells established from bone marrow samples from patients with relapsed/refractory, FLT3-ITD and/or complex karyotype AML (n=7) . As shown in Figure 9B, statistically significant inhibition of viable CFU was observed following combined IMGN779 (10 pM) and olaparib (1 μM) therapy compared to monotherapy or vehicle controls (p<0.001). These results suggest that the combination of IMGN779 with olaparib can be effective in clinical chemoresistant disease settings.

Example 4. Combinations of IMGN779 with niraparib and IMGN779 with taprazopanib show enhanced antileukemic activity, induce S-phase arrest, and increase apoptosis and DNA damage in vitro

Human CD33+ AML cell lines (HEL-luc and HL60) were treated in vivo with different dose ranges (100 pM-1 nM) of IMGN779 alone and in combination with each of the following PARP inhibitors: lucaparib, veliparib, Talazopanib and Niraparib. Proliferation was measured after incubation with WST-8 reagent. Synergistic/additive effects were calculated at different drug concentrations using Compusyn software. Flow cytometry was also performed for apoptosis, survival, DNA damage/repair, and cell cycle effects following combination and single drug treatment.

Treatment with lucaparib, veliparib, niraparib, tarazopanib, and olaparib in HEL-luc and HL60 cell lines showed that tarazopanib was the most Potent PARP inhibitors (FIGS. 10A-10B, Table 3). Combination treatment with taprazopanib alone (0.8 μM) and in combination with IMGN779 (800 pM) resulted in an increase in fractional cell survival in the same cell lines compared to combination treatment with olaparib and niraparib at the same concentrations. Maximum reduction (FIGS. 11A-11C). Combination indices of IMGN779+talazopanib and IMGN779+niraparib therapy calculated by Compusyn were less than 1, consistent with a synergistic effect (FIG. 12A-FIG. 12B). In addition, the combination of IMGN779 + tarazopanib at the tested concentrations resulted in apoptosis compared to the combination of IMGN779 + niraparib and IMGN779 + olaparib in the HEL-luc cell line (FIG. 13A-FIG. 13C), S Phase cell cycle arrest (FIG. 14A-FIG. 14C) and the greatest increase in DNA damage (FIG. 15A-FIG. 15C). The results of these experiments further support the mechanism of action of PARP inhibitors and support the use of PARP inhibitors in combination with IMGN779 for the treatment of cancer.

Table 3. IC50 values of PARP inhibitors in AML cell lines

Claims (35)

1. A method of treating cancer in a subject, comprising the steps of: administering to said subject an effective amount of a poly-ADP ribose polymerase (PARP) inhibitor and an effective amount of an antibody of formula (I)- drug conjugate or a pharmaceutically acceptable salt thereof, wherein: Double line between N and C represents a single bond or a double bond, provided that when the double bond is a double bond, X is absent and Y is hydrogen; when the double bond is a single bond, X is hydrogen and Y is -SO ₃ H; wherein A is an antibody or antigen-binding fragment thereof that specifically binds to CD33, which comprises the heavy chain variable region (VH) complementarity determining region (CDR) 1 sequence of SEQ ID NO:1, the VH CDR2 sequence of SEQ ID NO:2 and the VH CDR3 sequence of SEQ ID NO:3, and the light chain variable region (VL) CDR1 sequence of SEQ ID NO:4, the VL CDR2 sequence of SEQ ID NO:5, and the VL CDR3 sequence of SEQ ID NO:6; and r is an integer from 1 to 10. 2. The method of claim 1, wherein the antibody-drug conjugate is ADC1 or a pharmaceutically acceptable salt thereof. 3. The method of claim 1, wherein the antibody-drug conjugate is ADC2 or a pharmaceutically acceptable salt thereof. 4. The method of any one of claims 1-3, wherein the pharmaceutically acceptable salt is a sodium or potassium salt. 5. The method of any one of claims 1-4, wherein the PARP inhibitor is a PARP-1 inhibitor. 6. The method of any one of claims 1-4, wherein the PARP inhibitor is a PARP-2 inhibitor. 7. The method of any one of claims 1-6, wherein the PARP inhibitor is selected from the group consisting of: or a pharmaceutically acceptable salt thereof. 8. The method of claim 7, wherein the PARP inhibitor is or a pharmaceutically acceptable salt thereof. 9. The method of any one of claims 1-8, wherein the antibody or antigen-binding fragment thereof comprises a recombinant protein comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7 or 9. chain variable region. 10. The method of any one of claims 1-8, wherein the antibody or antigen-binding fragment thereof comprises an amino acid sequence comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 8 or 10. chain variable region. 11. The method of any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO:9 and a sequence comprising SEQ ID NO:10 light chain variable region. 12. The method of any one of claims 1-11, wherein the antibody is huMy9-6. 13. The method of claim 12, wherein the antibody is a CDR grafted or resurfaced antibody. 14. The method of any one of claims 1-13, wherein the antibody-drug conjugate is IMGN779. 15. The method of any one of claims 1-14, wherein the cancer is selected from the group consisting of leukemia, lymphoma, and myeloma. 16. The method of claim 15, wherein the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), B cells Lineage acute lymphoblastic leukemia (B ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), myelodysplastic syndrome (MDS), basal plasmacytoid DC neoplasia (BPDCN) leukemia, non- Hodgkin's lymphoma (NHL), mantle cell lymphoma and Hodgkin's leukemia (HL). 17. The method of claim 16, wherein the cancer is acute myeloid leukemia (AML). 18. The method of claim 17, wherein the acute myeloid leukemia (AML) is refractory or relapsed acute myeloid leukemia. 19. The method of claim 17, wherein the acute myeloid leukemia (AML) is characterized by overexpression of P-glycoprotein; overexpression of EVI1; p53 changes; DNMT3A mutations; FLT3 internal tandem repeats; complex nuclei type; decreased expression of BRCA1, BRCA2, or PALB2; or mutations in BRCA1, BRCA2, or PALB2. 20. The method of any one of claims 15-19, wherein the cancer is chemotherapy-sensitive. 21. The method of any one of claims 15-20, wherein the cancer is chemotherapy resistant. 22. A pharmaceutical composition comprising: i) an effective amount of a PARP inhibitor; ii) an effective amount of an antibody-drug conjugate of formula (I): or a pharmaceutically acceptable salt thereof; and iii) a pharmaceutically acceptable carrier or diluent; wherein: Double line between N and C represents a single bond or a double bond, provided that when the double line is a double bond, X does not exist, and Y is hydrogen; when the double line is a single bond, X is hydrogen, and Y is -SO ₃ H; wherein A is an antibody or antigen-binding fragment thereof that specifically binds to CD33, which comprises the heavy chain variable region (VH) complementarity determining region (CDR) 1 sequence of SEQ ID NO:1, the VH CDR2 sequence of SEQ ID NO:2 and the VH CDR3 sequence of SEQ ID NO:3, and the light chain variable region (VL) CDR1 sequence of SEQ ID NO:4, the VL CDR2 sequence of SEQ ID NO:5, and the VL CDR3 sequence of SEQ ID NO:6; and r is an integer from 1 to 10. 23. The pharmaceutical composition of claim 22, wherein the antibody-drug conjugate is ADC1: or a pharmaceutically acceptable salt thereof. 24. The pharmaceutical composition of claim 22, wherein the antibody-drug conjugate is ADC2: or a pharmaceutically acceptable salt thereof. 25. The pharmaceutical composition of any one of claims 22-24, wherein the pharmaceutically acceptable salt is a sodium or potassium salt. 26. The pharmaceutical composition of any one of claims 22-25, wherein the PARP inhibitor is a PARP-1 inhibitor. 27. The pharmaceutical composition of any one of claims 22-25, wherein the PARP inhibitor is a PARP-2 inhibitor. 28. The pharmaceutical composition of any one of claims 22-27, wherein the PARP inhibitor is selected from the group consisting of: or a pharmaceutically acceptable salt thereof. 29. The pharmaceutical composition of claim 28, wherein the PARP inhibitor is or a pharmaceutically acceptable salt thereof. 30. The pharmaceutical composition of any one of claims 22-29, wherein the antibody or antigen-binding fragment thereof comprises an amino acid sequence comprising at least 95% identity to the amino acid sequence of SEQ ID NO: 7 or 9 heavy chain variable region. 31. The pharmaceutical composition of any one of claims 22-29, wherein the antibody or antigen-binding fragment thereof comprises an amino acid sequence comprising at least 95% identity to the amino acid sequence of SEQ ID NO: 8 or 10 light chain variable region. 32. The pharmaceutical composition of any one of claims 22-31, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO:9 and a heavy chain variable region comprising the sequence of SEQ ID NO:10 The sequence of the light chain variable region. 33. The pharmaceutical composition of any one of claims 22-32, wherein the antibody is huMy9-6. 34. The pharmaceutical composition of any one of claims 22-32, wherein the antibody is a CDR-grafted or resurfaced antibody. 35. The pharmaceutical composition of any one of claims 22-34, wherein the antibody-drug conjugate is IMGN779.